Viruses invade all forms of life, causing diseases in humans including HIV-AIDS. Although the variety of viruses is daunting, all enveloped viruses including those studied here, HIV-1 and Rous Sarcoma Virus (RSV), must associate with the cytoplasmic leaflet of plasma membranes. One difficulty for in vitro studies is the lack of good models of the plasma membrane cytoplasmic leaflet. Another difficulty is that real cells can have membrane heterogeneities on the tens of nanometer size scale on the opposed, exoplasmic leaflet. Both of these experimental issues are addressed in these proposed studies, with a multicomponent model cytoplasmic mixture that can be coupled to a phase-separated lipid mixture in an asymmetric bilayer. This project will explore how three aspects of membrane lipid mixing behavior are related to viral Gag protein binding and assembly: (1) How is the thermodynamic activity of membrane-bound phosphatidylserine controlled by lipid composition, and how is this PS activity connected to Gag binding? (2) How is the thermodynamic activity of Gag's other binding partner, PI(4,5)P2 controlled by the other membrane lipids, and in particular, what factors control the formation of PI(4,5)P2 domains? Are these domains the sites of Gag assembly? (3) The plasma membrane is asymmetric. How does the presence of a phase-separated leaflet that is coupled to the cytoplasmic leaflet change Gag binding and assembly? A theme of this work is that the tendency of membrane lipids to bind or react is described by their thermodynamic activity, and this activity is controlled by all the components of the mixture. This approach provides predictive power to describe the associations of viral Gag structural proteins with their lipid binding partners: Which membrane factors exert control over the interactions among membrane-bound viral Gag proteins? The overall strategy is to combine measurements of lipid thermodynamic activity with measurement of virus protein binding and assembly. Fluorescence microscopy is used to visualize domains of PI(4,5)P2, and to correlate these domains with measured Gag binding and Gag-Gag assembly into its viral lattice.